Mass spectrometric identification of all types of molecules relies on the observation and interpretation of ion fragmentation patterns. Peptides, proteins, carbohydrates and nucleic acids that are often found as components of complex biological samples represent particularly important challenges. The most common strategies for fragmenting biomolecular ions include low- and high-energy collisional activation, post-source decay, and electron capture or transfer dissociation. Each of these methods has its own idiosyncrasies and advantages but encounters problems with some types of samples. Novel fragmentation methods that can offer improvements are always desirable. One approach that has been under study for years but is not yet incorporated into a commercial instrument is ultraviolet photofragmentation. This review discusses experimental results on various biological molecules that have been generated by several research groups using different light wavelengths and mass analyzers. Work involving short-wavelength vacuum ultraviolet light is particularly emphasized. The characteristics of photofragmentation are examined and its advantages summarized.
One Hundred Fifty-Seven nm photodissociation of singly protonated peptides generates unusual distributions of fragment ions. When the charge is localized at the C-terminus of the peptide, spectra are dominated by x-, v-, and w-type fragments. When it is sequestered at the N-terminus, a-and d-type ions are overwhelmingly abundant. Evidence is presented suggesting that the fragmentation occurs via photolytic radical cleavage of the peptide backbone at the bond between the ␣-and carbonyl-carbons followed by radical elimination to form the observed daughter ions. Low-energy fragmentation appears to be well described by the mobile proton model according to which vibrational excitation of the analyte leads to charge mobility [12]. Transfer of the charge proton to either the backbone carbonyl oxygen or amide nitrogen enables charge-induced cleavage of the peptide backbone. This process yields primarily b-and y-type fragment ions according to the standard nomenclature shown below [13,14].Higher energy activation methods involving collisions with gas molecules or surfaces can enable chargeremote fragmentation [15][16][17]. However, even in these cases, apparently, protonated peptides still generate some fragments through charge-directed processes [17]. Immobilization of the charge, either by using metal adducts [18] or charged chemical modifications [19 -22], has been used to inhibit charge-directed fragmentation. In these cases, primarily a-, d-, and w-type fragments are observed. Several mechanisms have been proposed for charge-remote peptide fragmentation, some involving homolytic radical cleavage [14,23].Electron capture dissociation (ECD) [24] and the recently reported electron-transfer dissociation (ETD)[25] appear to be nonthermal processes. These phenomena generate c-and z-type fragment ions upon the addition of an electron to a multiply charged protein or peptide ion. Two mechanisms have been proposed, one involving reactions of a free hydrogen atom generated when an electron is captured by a charged site on the analyte ion [26], and the other involving localization of the ϳ6 eV of energy that is generated upon charge neutralization. This energy then induces fragmentation through an excited electronic state [27]. Implicit in this second mechanism is the suggestion that techniques which excite appropriate electronic states can lead to unique, nonergodic fragmentation even for molecules as large as peptides and proteins [24,27]. This can occur if ions reach a dissociative electronic state and fragment before the energy is redistributed throughout the molecule. Electronic to vibrational relaxation, resulting in nonspecific excitation is an important competing process that often inhibits nonergodic processes, and rates of relaxation are predicted to increase with the size of the molecule [28].Lasers are powerful tools that can also be used to excite molecules to specific energy levels with either multiphoton or single photon processes. Light is not affected by the electric or magnetic fields of mass spectrometers, s...
We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a protein's quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.
Improved procedures for guanidination of lysine-containing peptides, a derivatization that results in increased MALDI mass spectral signal intensities are presented. The complete conversion of lysines to homoarginines can be accomplished in as little as 5 min. The method is demonstrated on a model peptide and on tryptic digests of three proteins. To demonstrate the applicability to proteomics samples, it is successfully applied to the digest of 50 fmol of a protein. Approaches for concentrating and purifying low-quantity protein digests following guanidination are evaluated. Experiments with the model peptide GRGDSPK enable investigation of the specificity of the guanidination reaction.
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